PROJECT SUMMARY/ ABSTRACT Background: Maternal hyperglycemia is a well-recognized risk factor for fetal congenital heart disease. However, the underlying cellular and molecular mechanisms are not well characterized. I hypothesize that maternal hyperglycemia leading to congenital heart defects and cardiac dysfunction are linked to abnormal DNA methylation and mRNA expression at cardiac specific loci. Methods: Hyperglycemia was induced in normal 8-week old CD-1 female mice with a one-time intraperitoneal injection of 150 mg/kg of streptozotocin (STZ) 2 weeks prior to mating. Histological analysis of fetal cardiac morphology was evaluated for malformations on embryonic day (E) 16.5 of control pups and pups exposed to maternal hyperglycemia. High frequency echocardiograms were performed on the adult progeny exposed to maternal hyperglycemia and control and 8 months of age. We used a massively-parallel sequencing-based methylation sensitive restriction based assay to examine genome-wide cytosine methylation levels at >1.65 million loci in neonatal hearts on post-natal (P) day 0. Functional validation was performed with real time quantitative polymerase chain reaction (RT-qPCR). Results: Cardiac structural defects occurred in 28% of the pups (n=12/45) of hyperglycemic dams versus 8% (n=7/87) of controls. Notable phenotypes were hypoplastic left or right ventricle, double outlet right ventricle, ventricular septal defect, and left ventricular outflow tract obstruction. Adult female progeny exposed to maternal hyperglycemia demonstrated reduced heart volumes on echocardiograms and increased fibrosis on histological evaluation. A 10-fold increase in DNA methylation of gene promoter regions was seen in many cardiac important genes in the experimental versus control P0 neonates and have corresponding decreases in gene expression in 21/32 genes functionally validated. Conclusion: Maternal hyperglycemia alters DNA methylation and mRNA expression of some cardiac genes during heart development. Quantitative, genome-wide assessment of cytosine methylation can be used as a discovery platform to gain insight into the mechanisms of hyperglycemia-induced cardiac anomalies. 1